car and pxr increase insig-1 expression - Molecular Pharmacology

Molecular Pharmacology Fast Forward. Published on February 21, 2008 as doi:10.1124/mol.107.041012 

MOL Manuscript #41012 

REGULATORY CROSSTALK BETWEEN DRUG 

METABOLISM AND LIPID HOMEOSTASIS: CAR AND PXR 

INCREASE INSIG-1 EXPRESSION 

Adrian Roth, Renate Looser, Michel Kaufmann, Sharon Blaettler, Franck Rencurel, Wendong 

Huang, David D. Moore 

and Urs A. Meyer 

Division of Pharmacology/Neurobiology, Biozentrum of the University of Basel, Klingelbergstrasse 

50-70, CH-4056 Basel, Switzerland (AR, RL, MK, SB, FR, UAM); Department of Gene Regulation 

& Drug Discovery, Beckman Research Institute, City of Hope National Medical Center, 1500 East 

Duarte Road, Duarte, CA 91010, USA (WH); Department of Molecular and Cellular Biology, Baylor 

College of Medicine, One Baylor Plaza, Houston, TX 77030, USA (DDM) 

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. 

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Running Title: CAR and PXR induce Insig-1 

Corresponding Author: Urs A. Meyer 

Number of Text Pages : 25 

Number of Tables : 1 

Number of Figures : 6 

Number of References : 31 

Division of Pharmacology/Neurobiology 

Biozentrum of the University of Basel 

Klingelbergstrasse 50-70 

CH-4056 Basel 

Switzerland 

Phone +41 61 267 22 20 

Fax +41 61 267 22 08 

Number of words in the Abstract: 247 

Number of words in the Introduction: 437 

Number of words in the Discussion: 974 

Email Urs-A.Meyer@unibas.ch 

Nonstandard abbreviations used : CAR, constitutive androstane receptor; CYP, cytochromes P450; 

ER, endoplasmatic reticulum, HA, hemagglutinin; PB, Phenobarbital; 

PCN, pregnenolone-16α-carbonitrile; PXR, pregnane x receptor; 

TCPOBOP, 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene; AICAR, 5- 

aminoimidazole-4-carboxamide riboside 

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ABSTRACT 


Activation of PXR (pregnane x receptor) and CAR (constitutive androstane receptor) by xenobiotic 

inducers of cytochromes P450 is part of a pleiotropic response that includes liver hypertrophy, tumor 

promotion, effects on lipid homeostasis and energy metabolism. Here we describe an acute response 

to CAR and PXR activators that is associated with induction of Insig-1, a protein with antilipogenic 

properties. We first observed that activation of CAR and PXR in mouse liver results in activation of 

Insig-1 along with reduced protein levels of the active form of sterol regulatory element binding 

protein 1 (Srebp-1). Studies in mice deficient in CAR and PXR revealed that the effect on 

triglycerides involves these two nuclear receptors. Finally, we identified a functional binding site for 

CAR and PXR in the Insig-1 gene by in vivo, in vitro and in silico genomic analysis. Our experiments 

suggest that activation of Insig-1 by drugs leads to reduced levels of active Srebp-1 and consequently 

to reduced target gene expression including the genes responsible for triglyceride synthesis. The 

reduction in nuclear Srebp-1 by drugs is not observed when Insig-1 expression is repressed by siRNA. 

In addition, we observed that Insig-1 is also a target of AMP-activated kinase (AMPK), the hepatic 

activity of which is increased by activators of CAR and PXR, and which is known to cause a 

reduction of triglycerides. The fact that drugs which serve as CAR or PXR ligands induce Insig-1 

might have clinical consequences and explains alterations in lipid levels after drug therapy. 

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Induction of cytochromes P450 (CYPs), other drug-metabolizing enzymes and drug transporters by 

their own substrates and other chemicals is an adaptive response of the liver to prevent accumulation 

of toxic xenobiotics and endobiotics. Xenosensors that mediate this response are the nuclear receptors 

pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR; for a review see 

(Handschin and Meyer, 2003)). PXR and CAR form heterodimers with the retinoid X receptor (RXR) 

and bind to specific DNA sequences in the regulatory region of target genes. PXR and CAR induce an 

overlapping set of genes involved in metabolism and transport of drugs (Maglich et al., 2002) but also 

genes involved in the regulation of steroids, bile acids, eicosanoids and genes involved in cholesterol 

and bile acid homeostasis (Huang et al., 2003; Staudinger et al., 2001) . 

Insig-1 and Insig-2 are proteins of endoplasmatic reticulum (ER) membrane and play an important 

role in the control of triglyceride and cholesterol biosynthesis (Yabe et al., 2002; Yang et al., 2002). 

The two isoforms bind in a sterol-dependent fashion to another ER membrane protein, sterol 

regulatory element binding protein (Srebp) cleavage-activating protein, or Scap, a transport protein 

needed for escort and subsequent activation of Srebp transcription factors (Hua et al., 1996). When 

Insig proteins are activated by sterols, insulin or other stimuli, they retain the Scap-Srebp complex in 

the ER membrane thereby preventing Srebp-dependent target gene expression. Srebp’s are a group of 

basic helix loop helix transcription factors, which activate an array of genes involved in the synthesis 

of cholesterol and triglycerides. While Srebp-2 is mainly involved in cholesterol biosynthesis, Srebp- 

1a and Srebp1c mainly activate genes involved in fatty acid and triglyceride synthesis (Shimano, 

2001). 

A decrease in hepatic and/or serum lipids, in particular triglycerides, has been observed in rodents 

after treatment with inducers of xenobiotic metabolism many years ago (Bjondahl, 1978; Hall et al., 

1990; Venkatesan et al., 1994). More recently, known inducers of human drug metabolism such as the 

commonly used anti-retroviral drug efavirenz or the barbiturate phenobarbital (PB) also have been 

shown to inhibit lipogenesis (Hadri et al., 2004; Kiyosawa et al., 2004). The molecular mechanism of 

the effect of inducers on triglycerides has not been explained. 

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In the present study we show that nuclear receptors CAR and PXR transcriptionally activate Insig-1 

by binding to an enhancer sequence of the Insig-1 gene. Our results explain the negative effect of 

drugs and xenobiotics on hepatic lipids in vivo and show that CAR and PXR not only play a role in 

the catabolism of various endogenous and exogenous compounds but also directly affect lipogenic 

pathways by activating Insig-1. Moreover, as Insig-1 has recently been found to be a possible drug 

target for the treatment of diabetes (Nakagawa et al., 2006), this study contributes to the 

understanding of the regulation of this gene and possibly to the development of new therapies against 

dislipidemia. 

5

MATERIALS & METHODS 

Animals 


C57BL/6 mice were maintained in a 12-hour light/12-hour dark cycle and had free access to food and 

drinking water. 9-11 week old male animals received an i.p. injection of either 100 mg/Kg 

phenobarbital (PB, Sigma, Buchs, Switzerland), 10 mg/Kg 1,4-Bis[2-(3,5- 

dichloropyridyloxy)]benzene (TCPOBOP; Bayer, Leverkusen, FRG) or 40 mg/Kg pregnenolone-16α- 

carbonitrile (PCN; Sigma, Buchs, Switzerland) in a 5% DMSO-corn oil solution i.p. or with vehicle 

10h before dissection. Animals were sacrificed by exposure to CO2, blood was collected by heart 

puncture, livers excised and snap-frozen in liquid nitrogen and stored at -80°C until use. 

Analysis of Triglycerides and Cholesterol 

50-100 mg of liver was used for each preparation. After weight determination, liver samples were put 

in an ethanol: ether (3:1, v/v) mixture in Fastprep tubes (Lysing matrix D, Qbiogene, Basel, 

Switzerland). Livers were homogenized on the Fastprep instrument for 40s at position 6,5 and 

evaporated to complete dryness on a Speed-vac evaporator. Samples were redissolved in 1ml 

isopropanol and tissue remnants spun down for 5 minutes at 14000 xg. 600 µl of the supernatant was 

mixed with 400 µl of water and lipids were determined using the esterase/oxidase kit for cholesterol 

determination and the L-α-glycerol phosphate oxidase kit for the determination of triglycerides 

(Roche Diagnostics, Rotkreuz, Switzerland). 

RT PCR Analysis 

RNA from cells and tissues was isolated using Tri-Reagent (Sigma, Buchs, Switzerland). One 

microgram total RNA was reverse-transcribed with MMLV reverse transcriptase (Roche Molecular 

Biochemicals, Rotkreuz, Switzerland). PCR was performed using the TaqMan PCR Core Reagent Kit 

(PE Applied Biosystems, Rotkreuz, Switzerland) and the transcript level quantitated with an ABI 

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PRISM 7700 Sequence Detection System (PE Applied Biosystems, Rotkreuz, Switzerland) according 

to the manufacturer’s protocol. Briefly, relative transcript levels were determined using the relative 

quantitation method by measuring the ∆Ct between the gene of interest and the internal control 

GAPDH. Primers and fluorescent probes used in these PCRs are listed in Table 1. 

Reporter Gene Assays 

Culture and transfection of CV-1 cells with Lipofectamine Transfection Reagent (Invitrogen, 

Carlsbad, USA) was performed as previously published (Handschin et al., 2000). Expression vectors 

encoding mouse PXR and mouse CAR as well as the beta-galactosidase vector used for signal 

normalization have been described (Handschin et al., 2002). For construction of hemaglutinin-tagged 

vp16 fusion proteins nuclear receptor sequences were amplified from expression plasmids and 

subcloned into pcDNA3/vp16-HA (a kind gift from Dr. Dieter Kressler, Biozentrum, University of 

Basel, Switzerland). Reporter vectors were based on PGL3-LUC (Promega, Wallisellen, Switzerland). 

Genomic DNA from the murine Insig1 enhancer region was amplified using PCR primers carrying 

restriction sites suitable for direct subcloning into the reporter vector. 

Preparation of Primary Hepatocytes 

For the preparation of mouse hepatocytes animals were anaesthetized with Ketamine/Xylazine (Sigma, 

Buchs, Switzerland). The portal vein was canulated and perfused with HEPES-EGTA (pH 7.4) for 5 

minutes and then with collagenase (type 2, Worthington, Lakewood NJ, USA) for 6 minutes. The 

livers were excised, cells were filtered through a nylon mesh and centrifuged at 50 xg at 4°C for 5 

minutes three times. After determination of viability, cells were plated at a density of 400’000 

cells/well (12 well plate) and were allowed to attach for 2 hours in William’s E medium without 

phenol red (Invitrogen, Basel, Switzerland), 10 % FCS, 4µg/ml insulin, 200µM glutamine and 1% 

penicillin/streptomycin (50 IU/ml) on collagen-coated dishes. Induction experiments were performed 

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in the same medium without FCS and with reduced insulin (2 µg/ml) but with the addition of 1 µM 

hydrocortisone. 

Primary human hepatocytes in suspension were allowed to attach on collagen-coated plates in DMEM 

supplemented with 10% FBS, 1% penicillin/streptomycin (50 IU/ml) and 1 µM dexamethasone 

overnight before start of the experiments. For induction cells were cultured in DMEM without serum 

but supplemented with insulin-transferrine-selenium mixture (Sigma, Buchs, Switzerland) and 1µM 

hydrocortisone 

Production of recombinant adenovirus particles 

Expression cassettes of interest were PCR-amplified using vector-specific primers with attB1/attB2- 

Gateway extensions for subsequent cloning into pDONR221 (Invitrogen, Carlsbad, CA, USA). For 

pcDNA3 constructs (Vp16-PXR, Vp16-CAR) the following primers were used: 

TTAGGGTTAGGCGTTTTGCGC (Fwd), TCAGAAGCCATAGAGCCCAC (Rev). Entry clones 

carrying the PCR products were used for cloning into pAd-DEST (Invitrogen, Carlsbad, CA, USA). 

PacI-digested plasmids were transfected into HEK293A cells and adenovirus particles produced and 

processed according to the manufacturer’s recommendations (Invitrogen, Carlsbad, CA, USA). 

Functionality of PXR- and CAR-expressing adenoviruses was assessed in reporter gene assays in CV- 

1 cells using PXR- and CAR-responsive reporter vectors. We also tested the recombinant proteins in 

mouse hepatocytes by measuring mRNA expression of the target genes Cyp3a11 and Cyp2b10, 

respectively (data not shown).The adenovirus particles encoding recombinant forms of AMPK have 

been described (20) 

Immunoblotting, Gel-Mobility-Shift Assay & Chromatin Immunoprecipitation 

For Western blot analysis of SREBP1, liver proteins were extracted from 100-200 mg of frozen tissue 

in 1 ml ice-cold buffer (50 mM Tris-HCl pH 7.4, 100mM KCl, 1mM EDTA pH 8.0, 10mM beta- 

Mercaptoethanol, 5mM DTT, 0.1% (v/v) Triton X-100, 0.1% (v/v) NP 40, 1 tablet/50ml buffer 

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Protease Inhibitor Cocktail (Roche Diagnostics, Rotkreuz, Switzerland)) in a 5 ml polystyrene tube 

using a Polytron Rotor-Stator Homogenizer. The homogenate was centrifuged for 30 min at 10000 

rpm at 4°C and 50 µg of protein was loaded on a 10% SDS-gel. SREBP1 isoforms were detected 

using mouse anti-SREBP1 monoclonal antibody (Anti-SREBP1 monoclonal #557036, Clone IgG- 

2A4BD, Pharmingen, Allschwil, Switzerland). 

Transcription factors were synthesized in vitro by using the TNT T7 Quick Coupled 

Transcription/Translation System (Promega, Wallisellen, Switzerland) according to the 

manufacturer's instructions. Probes were labeled with the Klenow fragment of DNA polymerase in the 

presence of radiolabeled [ -32P]ATP, and the probe was purified over a Biospin 6 chromatography 

column. A volume of labelled oligonucleotide corresponding to 100,000 cpm was used for each 

reaction in 10 mM Tris HCl, pH 8.0/40 mM KCl/0.05% Nonidet P-40/6% (vol/vol) glycerol/1 mM 

DTT containing 0.2 µg of poly(dI-dC) poly(dI-dC) and 2.5 µl of the in vitro synthesized proteins as 

described previously (Handschin et al.). The mix was incubated for 20 min at room temperature and 

subsequently electrophoresed on a 6% polyacrylamide gel in 0.5× Tris/borate/EDTA buffer (1x TBE 

buffer is composed of 0.9 M Tris-Borate, 0.002 M EDTA, pH 8.3) followed by autoradiography at 

70°C. Oligos used for EMSAs were obtained as follows: For the mouse Insig-1 DR4 the following 

oligonucleotide were annealed and labelled using polynucleotide kinase: 

CCTGAGGGTCAACAGAGGACACCTAG (Fwd) and CTAGGTGTCCTCTGTTGACCCTCAGG 

(Rev). 

Chromatin Immunoprecipitation was performed using the EZ-Chip kit from Upstate 

(Charlottesville, USA). Primary mouse hepatocytes were infected with adenoviral particles encoding 

HA-tagged vp16-mouse CAR or mouse PXR, respectively. After 24 hours cells were harvested and 

samples processed according to the manufacturers recommendations. For immunoprecipitation 

hemagglutinin (HA) antibody (monoclonal HA.11 clone 16B12 mouse IgG1 MMS-101P) from 

Covance (Princeton, USA)was used. 

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Targeting of Insig-1 in primary mouse hepatocytes by siRNA 

For the transfection of primary mouse hepatocytes, Dharmafect1 transfection reagent (#T2001-01, 

Dharmacon, Chicago, USA) was used. 100nM siRNAs (siGenome SmartPool mINSIG1 #M-060068- 

00; siControl Nontargeting siRNAPool#2 #D-001206-14-05; Dharmacon, Chicago, USA) and 14µl 

Dharmafect were used according to the manufacturer’s instructions. 6h after transfection medium was 

removed and replaced with fresh medium without serum. 24h later, medium was replace by medium 

containing 500µM phenobarbital and mRNAs and Srebp-1 protein analyzed after 24h as described. 

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RESULTS 


Mice were injected with drugs known to activate nuclear receptors PXR and CAR, namely 

phenobarbital (PB, activator of both PXR and CAR), pregnenolone-16α-carbonitrile (PCN, activator 

of PXR) and 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP, activator of CAR; Fig. 1). 

After 10 hours of exposure, liver samples were analyzed for hepatic triglycerides and cholesterol (Fig. 

1, top panel). All three drugs caused a substantial drop in triglycerides, whereas cholesterol levels 

were less affected. PB and TCPOBOP at the doses applied resulted in a 49% or 67% decrease in 

triglycerides, respectively, and a 28% or 33% decrease in cholesterol. Serum analysis of PB-treated 

animals revealed no significant change in triglycerides or cholesterol at 10 hours (Fig. 1, top panel, 

right). RT-PCR analysis of these livers showed marked induction of Insig-1 mRNA (Fig. 1, middle 

panel). The mRNA levels of Insig-2 were not significantly induced by drug treatment (data not 

shown) and mRNA levels of Srebp genes remained unaffected by drug treatment as well. Accordingly, 

Hmg-CoA reductase (Hmgcr), a target gene of Srebp-2 (Horton et al., 1998) was unchanged while 

Stearoyl-CoA desaturase 1 (Scd-1), which is a target gene of Srebp-1 (Shimano et al., 1999), was 

reduced after drug treatment. Again, TCPOBOP showed strongest effects (Fig. 1, middle panel). 

Whether the reduction in hepatic triglycerides was due to reduced nuclear expression of Srebp-1 was 

tested by immunoblotting of liver protein extracts from drug-treated animals using an antibody which 

can discriminate between the inactive precursor form of Srebp1 and the activated nuclear (mature) 

form of Srebp-1 (Fig. 2A). The graph shows a reduction in nuclear content of Srebp-1 in drug-treated 

mice with strongest effects by PB and TCPOBOP. A time course experiment in primary mouse 

hepatocytes revealed that the time-dependent induction profile of Insig-1 mRNA paralleled the one of 

the classic CAR- and PXR-inducible gene Cyp2b10 (Fig. 2B). 

To define the role for PXR and CAR in the activation of Insig1 and the subsequent drop in hepatic 

triglycerides we applied PB to mice deficient in these two receptors (Zhang et al., 2004), Fig. 3). Fig. 

3 shows that two typical target genes of PXR and CAR, Cyp2b10 and Cyp3a11, expectedly were not 

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inducible by PB in PXR/CAR null mice. Blunted induction of Insig-1 mRNA in these animals after 

PB treatment as well as an unchanged triglyceride profile compared to wild type animals was 

observed (Fig. 3, lower panels). These data support a PXR/CAR-dependent mechanism for the 

triglyceride-lowering effect of inducer compounds. 

We therefore designed experiments to identify functional binding sites for the PXR and CAR in the 

regulatory region of Insig-1. Two 3kb fragments of genomic DNA from the 5’-flanking region of the 

Insig1 gene were cloned into a luciferase reporter vector and activity was assessed in transactivation 

assays in CV-1 cells (Fig. 4A). The first DNA element spanned the transcriptional start site including 

the proximal promoter of Insig1 to -3044 bp and showed no activation after drug treatment. The 

second large DNA stretch was overlapping with the first one and ended at –6252bp. There was slight 

(as compared to empty control luciferase vector) activation of reporter gene transcription after drug 

treatment. This DNA element was cut into smaller pieces and activity assessed until a 760 bp 

fragment revealed a robust response to PXR and CAR. Within this fragment a DR-4 type drug 

response element was identified using the Nubiscan algorithm (Podvinec et al., 2002). This element 

responded well to PXR and CAR and specificity was assessed using a mutated version of the DR-4 

element, which resulted in a decreased response to drugs (Fig. 4A). The same DR-4 element was used 

in electromobility shift assays together with in vitro translated PXR, CAR and their heterodimeric 

partner, RXR (Fig. 4B). A strong band was observed when CAR and RXR were incubated with the 

oligonucleotide carrying the DR-4 element and less intensive binding appeared when PXR was used. 

Both nuclear receptor complexes were super-shifted by co-incubation with an anti-RXR antibody. 

Functionality of this element was tested in vivo by chromatin immunoprecipitation in primary mouse 

hepatocytes (Fig. 4C). Cells were infected with adenovirus particles expressing HA-tagged versions 

of both CAR and PXR. The amplified PCR product corresponds to the 157bp region in the murine 

Insig-1 promoter where the designated DR-4 element is located. In Fig 5B a reporter gene analysis 

using the Insig-1 DR-4 with inducers of mouse PXR and CAR, respectively, was performed and 

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revealed a 4-fold induction after PCN-treatment and a 7.2-fold induction after TCBOBOP-treatment 

(Fig. 5A). 

A role for Insig-1 in mediating the repressive effects of inducer drugs was established by siRNA- 

mediated inhibition of Insig-1 expression and concurrent treatment with PB (Fig. 5B). Primary mouse 

hepatocytes were transfected with either unspecific control siRNAS or siRNA targeting Insig-1. After 

48h, mRNA analysis revealed markedly reduced Insig-1 expression (Fig. 5B, left panel). Immunoblot 

analysis of Srebp-1 protein levels in siRNA transfected cultures treated with or without PB was 

performed (Fig. 5B, right panel). The results reveal reduced nuclear expression of Srebp-1 in cells 

transfected with control siRNA and treated with PB. In cells where Insig-1 expression was reduced by 

siRNA, Srebp-1 protein levels in the nucleus remained unaffected (Fig. 5B, right panel). 

To reveal a potential role of this mechanism in humans we tested the inducibility of human Insig1 by 

PB in primary human hepatocytes (Fig. 6A). Treatment for 50 hours with PB resulted in a significant 

induction of Insig-1 in cultures of two different donors and this was paralleled by a reduction in 

Srebp1c expression. Induction of CYP2B6 and CYP3A4 served as positive controls. 

As recently reported, induction of drug metabolizing enzymes requires activation of AMP-activated 

kinase (Rencurel et al., 2006; Rencurel et al., 2005). We therefore wanted to test whether this kinase, 

alone or in combination with drug, can regulate transcription of Insig1 (Fig. 6B). Primary human 

hepatocytes were infected with control virus (expressing beta-galactosidase) or different versions of 

AMPK: a dominant negative construct (kinase dead, KD), the alpha-1 or the alpha-2 subunit of 

AMPK. While the dominant negative version repressed expression of Insig1, both subunits induced 

Insig1 with stronger effects seen using the alpha-1 subunit. Furthermore, exposure to PB enhanced 

these effects (Fig. 6B). 

13

DISCUSSION 


The experiments described here reveal a novel molecular mechanism by which drugs that induce 

drug-metabolizing enzymes and drug transporters can acutely regulate hepatic triglyceride levels. 

While several CYPs and other enzymes involved in metabolism and transport of xenochemicals have 

been known to be targets of nuclear receptors CAR and PXR, the upregulation of Insig-1 by the same 

receptors after drug treatment is new and adds a potentially clinically important aspect to the present 

understanding on how the liver reacts to accumulating lipophilic compounds such as PB. This report 

shows that the drug metabolizing process also includes direct regulation of hepatic lipid biosynthesis 

by induction of an important regulatory protein as is Insig-1. We present evidence for a functional 

DR-4 binding site for CAR and PXR in the upstream promoter region of Insig-1 (Fig. 4). Binding of 

the xenobiotic receptors to this DR-4 site can account for the induction of Insig-1, which results in the 

reduced expression of the activated nuclear form of Srebp-1 and the substantial reduction in hepatic 

triglycerides after only 10 hours of treatment (Fig. 1,2). The fact that Insig-1 has been observed in 

expression studies to be induced early after treatment with the CAR ligand TCPOBOP (Locker et al., 

2003) and that overexpression of Insig-1 in livers of mice has been shown to cause a drop in 

triglyceride levels with smaller effects on cholesterol (Engelking et al., 2004; Takaishi et al., 2004) 

made Insig-1 an interesting candidate gene for transcriptional regulation by CAR and PXR. We first 

established that in CAR/PXR double knockout mice PB had lost its effect on triglycerides and there 

was no induction of Insig-1 (Fig. 3). This strongly supported the idea of a CAR/PXR-mediated 

transcriptional activation of the Insig-1 gene. Moreover, the induction of Insig1 mRNA appeared early 

after addition of drug (Fig. 2B) making a rapid decrease in activated nuckear form of Srebp-1 protein 

levels a plausible scenario (Fig. 2A). The more pronounced effects on triglycerides seen with the CAR 

activators TCPOBOP and PB compared to the PXR ligand PCN is in line with the more pronounced 

activation of the Insig1 promoter by CAR (Fig. 4A, 5B), with the higher affinity of CAR to bind to 

the DR-4 element (Fig. 4B) as well as with the stronger enrichment in CAR-immunoprecipitated 

14


samples of this fragment (Fig. 4C). A role for Insig-1 in mediating these effects was established in 

mouse hepatocytes with siRNA-reduced Insig-1 expression (Fig. 5B). While the repressive effect of 

PB on nuclear protein levels of Srebp-1 was evident, in cells with repressed Insig-1 expression this 

effect was not detectable, strongly supporting the concept that PXR/CAR-activated Insig-1 is 

responsible for reduced Srebp-1 and thereby for lowered hepatic triglycerides. Moreover, the fact that 

these effects could be reproduced in human hepatocytes supports the concept of a general mechanism 

by which drugs affect hepatic lipid biosynthesis (Fig. 6A). Insig- 2, the other member of the Insig 

family of Srebp-regulating genes was not affected by PXR/CAR inducers and a potential role of this 

gene in linking drug treatment to reduced triglyceride levels was not pursued. It cannot be ruled out of 

course, that under different conditions, this gene may also play a role in mediating lipid synthesis due 

to a xenobiotic challenge. 

An effect of PXR on lipogenic genes has recently been described by Nakamura et al. (Nakamura et 

al., 2007). In strong support of our data they observed downregulation of lipogenic genes by the PXR- 

specific activator PCN and this effect was not seen in PXR -/- mice. Interestingely, Nakamura and 

colleagues furthermore observed an induction of Scd-1 in PCN-treated animals that were fasted for 

24h. This suggests an additional level of regulation of lipogenesis by xenobiotics in the fasted state. 

Another new finding of our study is the role of AMPK in the induction of Insig1 (Fig. 6B). AMPK is 

considered a metabolic master-switch sensing cellular energy levels and regulating glucose transport 

and gluconeogenesis. It is activated in response to metabolic stress signals that deplete cellular ATP 

and stimulate fatty acid oxidation (Kahn et al., 2005). It was recently shown that CAR-dependent 

induction of CYP2B by PB requires activation of AMPK (Rencurel et al., 2006). Blattler et al. 

(Blattler et al., 2007) demonstrated that PB interferes with mitochondrial function and activates the 

AMPK upstream kinase LKB1 which then mediates the activation cascade of AMPK to CAR. 

Interestingly, AMPK, either via activation by AICAR (5-aminoimidazole-4-carboxamide riboside) or 

via adenoviral overexpression of its catalytic subunit, also has been shown to reduce Srebp-1c 

expression (Foretz et al., 2005; Zhou et al., 2001). As these observations lacked a mechanistic 

15


explanation, the activation of Insig-1 by AMPK shown here may indicate a signaling pathway leading 

to repression of Srebp-1c. In line with observations by Rencurel et al. (2006), AMPK alone seems 

capable of regulating expression of CAR/PXR target genes and addition of a nuclear receptor 

activator leads to a synergistic effect on gene transcription. However, the detailed interplay between 

PB, nuclear receptors and AMPK in the induction of Insig-1 clearly requires further investigation. 

Also, the data presented here account for the immediate physiologic response of the liver to a 

xenobiotic challenge. Chronic treatment with drugs leading to constant activation of PXR and/or CAR 

may lead to diverse adaptive gene-regulations to maintain lipid homeostasis (Kiyosawa et al., 2004; 

Zhou et al., 2006). 

In conclusion, the results of our experiments suggest that the signaling pathways involved in 

mediating the effect of xenobiotics on detoxification also induce Insig-1, a gene regulating lipid 

biosynthesis and that this is associated with an acute lowering of triglyceride levels in the liver. As 

Insig-1 has recently been suggested as a possible drug target for the treatment of dislipidemic diseases 

including diabetes (Nakagawa et al., 2006) the observation that CAR and PXR ligands or activators 

induce Insig-1 may have clinical consequences and explains the reported alterations in lipid levels 

after drug therapy. 

16

ACKNOWLEDGEMENTS 


The authors would like to thank Michael Podvinec (Institute of Bioinformatics, University of Basel, 

Switzerland) for assistance in in silico sequence analysis and Frederic Delobel (Hoffmann-La Roche 

Ltd, Basel, Switzerland) for experimental support with primary mouse hepatocytes. 

17

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20

FOOTNOTES 


This work was supported by grants of the Swiss National Science Foundation and the STEROLTALK 

project (EC contract No. LSHG-CT-2005-512096) under the 6 th framework program. 

21

LEGENDS TO FIGURES 

Fig. 1 


Lipid and gene expression changes after drug application in mouse liver. PB, TCPOBOP and 

PCN were i.p.-injected in mice and livers or serum samples analyzed after 10h. Upper panel, 

triglyceride (TG) and total cholesterol (CHOL) analysis in liver homogenates and blood samples. 

Middle and lower panel, RT-PCR analysis of RNAs from mouse livers. Bars indicate mean values 

from 6 animals per treatment group and standard deviations therefrom. *p

Fig. 4 


Analysis of the Insig1 promoter for drug response elements. A, Reporter gene assays using 

different genomic DNA sequences from the Insig1 promoter cloned into tk-Luc vector. At the bottom 

of the Fig. a Luc vector with a mutated Insig1 DR-4 was used.. Plasmids were cotransfected together 

with mouse CAR or PXR into CV-1 cells and luciferase activities determined 16h after addition of 

1µM TCPOBOP and 10µM PCN, respectively. Cell lysates were analyzed for luciferase expression 

and fold activation of reporter fragment calculated relative to empty luciferase vector treated with 

drugs is indicated. The sequence of the identified DR-4 type drug response element as well as the 

mutated form used in the reporter assay is shown in a separate box. B, Gel shift assay using in vitro 

translated mouse CAR, PXR and RXR and a radiolabelled oligonucleotide encoding the putative 

Insig1 DR-4 element. The experimental conditions for each lane are indicated in the panel above the 

picture: “+” indicates that the corresponding receptor was added, while “-“ indicates that no receptor 

was added , * position of shifted nuclear receptor heterodimer, ** position of anti-RXR supershifted 

nuclear receptor heterodimer. C, Association of CAR and PXR with the Insig1 DR-4 analyzed by 

chromatin immunoprecipitation in primary mouse hepatocytes using adenovirus encoding HA-tagged 

CAR and PXR, respectively. Lower panel shows a control experiment using the same lysates on the 

GAPDH promoter. 

androstanol (AND), 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and pregnenolone-16α- 

carbonitrile (PCN) 

Fig. 5 

Induction of Insig1 DR-4 reporter gene by PXR and CAR inducers and lack of effect of 

Phenobarbital on SREBP-1 proteins when Insig-1 expression is suppressed by RNAi. A, Effect 

of PXR, CAR and their ligands/activators on the Insig-1 DR-4 reporter gene. PXR was activated by 

over night treatment with 10µM pregnenolone-16α-carbonitrile (PCN). Constitutive active CAR was 

23


repressed by the addition of 1µM androstanol and activated by addition of 10µM 1,4-Bis[2-(3,5- 

dichloropyridyloxy)]benzene (TCPOBOP). Fold activations are calculated as relaitve reporter gene 

levels over non-drug-treated cells. B, primary mouse hepatocytes were transfected with either control 

siRNA or siRNA against Insig-1. mRNA analysis of Insig-1-siRNA effect after 48h is shown in the 

left panel, effect of PB treatment on Srebp-1 protein levels in cultures transfected with either control 

or Insig-1 siRNA is shown in the right panel. Asterisks indicate precursor (*) and mature form (**) of 

Srebp-1. 

Fig. 6 

Induction of Insig1 by PB and AMPK in primary human hepatocytes. A, Cells from two different 

donors were incubated with 500µM PB and mRNA analyzed 50h thereafter. B, AMPK and PB 

synergistically activate human INSIG-1. Primary human hepatocytes from a third donor were infected 

with adenoviral particles encoding either control gene (B-Gal), a dominant negative form of AMPK 

(KD), the alpha-1 subunit of AMPK (AMPKα1) or the alpha-2 subunit of AMPK (AMPKα2). 

*p


Table 1: Primers and Probes used for RT-qPCR 

Gene Forward Reverse Taqman-Probe 

Mouse: 

Gapdh CCAGAACATCATCCCTGCATC GGTCCTCAGTGTAGCCCAAGAT CCGCCTGGAGAAACCTGCCAAGTATG 

Cyp2b10 CAATGTTTAGTGGAGGAACTGCG CACTGGAAGAGGAACGTGGG CCCAGGGAGCCCCCCTGGA 

Cyp3a11 AGAACTTCTCCTTCCAGCCTTGTA GAGGGAGACTCATGCTCCAGTTA CTAAAGGTTGTGCCACGGGATGCAGT 

Srebp1c GGAGCCATGGATTGCACATT CCTGTCTCACCCCCAGCATA CAGCTCATCAACAACCAAGACAGTGACTTCC 

Scd-1* CCGGAGACCCTTAGATCGA TAGCCTGTAAAAGATTTCTGCAAACC 

Hmgcr* CGAGGAAAGACTGTGGTTTGTG CGTCAACCATAGCTTCCGTAGTT 

Insig-1* TGCAGATCCAGCGGAATGT CCAGGCGGAGGAGAAGATG 

Human: 

CYP2B6 ACATCGCCCTCCAGAGCTT GTCGGAAAATCTCTGAATCTCATAGA ACCGAGCCAAAATGCCATACACAGAGG 

CYP3A4 CATTCCTCATCCCAATTCTTGAAGT CCACTCGGTGCTTTTGTGTATCT CGAGGCGACTTTCTTTCATCCTTTTTACAGATTTTC 

INSIG-1* AGCCCCTACCCCAACACCT ACCACCCCAACCGAGAAGA 

SREBP1c* TCAGCGAGGCGGCTTTGGAGCAG CATGTCTTCGATGTCGGTCAG 

18s AGTCCCTGCCCTTTGTACACA CGATCCGAGGGCCTCACTA CGCCCGTCGCTACTACCGATTGG 

(*SYBR primers) 

25

mg/g Tissue 

Rel mRNA Level 

16 

14 

12 

10 

8 

6 

4 

2 

0 

6 

5 

4 

3 

2 

1 

0 

Triglycerides 

* * 

Insig1 

** 

** 

** ** 

Ctrl 

PB 

PCN 

TCPOBOP 

mg/g Tissue 



5 

4 

3 

2 

1 

0 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

Cholesterol 

Srebp1c 

Scd1 

* 

* 

** 

*p < 0.05 

* * p < 0.005 

mg/dl 

Figure 1 

* 

* 

Serum Chemistry 

400 

TG 

350 

300 

250 

200 

150 

100 

50 

0 



6 Srebp2 

5 

4 

3 

2 

1 

0 

6 Hmgcr 

5 

4 

3 

2 

1 

0 

* 

CHOL

kDa 

150 

100 

75 

50 

Rel. mRNA Level 

A 

B 

15 

12 

9 

6 

3 

1 

DMSO PB 

Cyp2b10 

Insig1 

* 

** 

kDa 

150 

100 

75 

50 

DMSO PCN TCPOBOP 

0 1 2 3 4 5 

Figure 2 

* 

** 

hrs after 

addition 

of drug

Relative mRNA Level 


400 Cyp2b10 

350 

300 

250 

200 

150 

100 

50 

0 

8 

7 

6 

5 

4 

3 

2 

1 

0 

WT Double-KO 

** 


- + - + PB - + - + PB 

Insig1 Triglycerides 

WT Double-KO 

20 

18 

WT Double-KO 

** 

16 

14 

mg/g Tissue 

3 

2.5 ** 

Cyp3a11 

WT Double-KO 

1.5 

0.5 

0 

12 

10 

Figure 3 

2 

1 

8 

6 

4 

2 

0 

- + - + PB - + - + PB 

**

A 

RXR 

CAR 

PXR 

Anti-RXR 

kb from transcriptional start site 

7 6 5 4 3 2 1 

-3044 

-6252 

-5118 

-6252 -5881 

-6252 -5111 

-5871 -5111 

-5340 -5314 

-5340 -5314 

X 

DR4 

- + - - + + 

- - - ++ +- 

++ 

B C 

+ - ++ 

- 

- 

- - - - + - + 

** 

* 

Figure 4 

0 

vp16PXR 

vp16CAR 

0 1 2 3 4 5 6 

fold activation reporter fragment 

over empty reporter vector 

-5333 

-5319 

wt …CCTGAGGGTCAACAGAGGACACCTAG… 

mut …CCTGAGTTTAAACAGAGGACACCTAG… 

control 

CAR 

PXR 

Insig1-DR4 

No Ab 

Input 

GAPDH

A 

Fold induction reporter 

B 


8 

7 

6 

5 

4 

3 

2 

1 

0 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

Ctrl-siRNA 

mInsig1-DR4 

- 

+mPXR 

+mCAR 

** 

Insig1-siRNA 

- 

kDa 

150 

100 

75 

50 

Androstanol 

PCN 

Figure 5 

Androstanol/ 

TCPOBOP 

* 

- + - 

** 

+ PB 

Ctrl-siRNA Insig1-siRNA

A 



4 INSIG1 

Donor 1 Donor 2 

3 

2 

1 

0 

16 

12 

8 

4 

0 

B 


- + - + PB 

CYP2B6 


- + - + PB 

14 INSIG1 

12 

10 

8 

6 

4 

2 

0 

* 



1.25 SREBP1c 


1 

0.75 

0.5 

0.25 

400 

300 

200 

100 

** 

0 

CYP3A4 


B-Gal KD AMPKalpha1 AMPKalpha2 

- - + - + - + PB 

Figure 6 

- + - + PB 

- + - + PB 

* 

**

car and pxr increase insig-1 expression - Molecular Pharmacology

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